Small Molecule-Induced\nPost-Translational Acetylation\nof Catalytic Lysine of Kinases in Mammalian Cells

Reversible lysine acetylation is\nan important post-translational modification (PTM). This process in\ncells is typically carried out enzymatically by lysine acetyltransferases\nand deacetylases. The catalytic lysine in the human kinome is highly\nconserved and ligandable. Small-molecule strategies that enable post-translational\nacetylation of the catalytic lysine on kinases in a target-selective\nmanner therefore provide tremendous potential in kinase biology. Herein,\nwe report the first small molecule-induced chemical strategy capable\nof global acetylation of the catalytic lysine on kinases from mammalian\ncells. By surveying various lysine-acetylating agents installed on\na promiscuous kinase-binding scaffold, <b>Ac4</b> was identified\nand shown to effectively acetylate the catalytic lysine of >100\ndifferent protein kinases from live Jurkat/K562 cells. In order to\ndemonstrate that this strategy was capable of target-selective and\nreversible chemical acetylation of protein kinases, we further developed\nsix acetylating compounds on the basis of VX-680 (a noncovalent inhibitor\nof AURKA). Among them, <b>Ac13</b>/<b>Ac14</b>, while\ndisplaying excellent in vitro potency and sustained cellular activity\nagainst AURKA, showed robust acetylation of its catalytic lysine (K162)\nin a target-selective manner, leading to irreversible inhibition of\nendogenous kinase activity. The reversibility of this chemical acetylation\nwas confirmed on <b>Ac14</b>-treated recombinant AURKA protein,\nfollowed by deacetylation with SIRT3 (a lysine deacetylase). Finally,\nthe reversible <b>Ac13</b>-induced acetylation of endogenous\nAURKA was demonstrated in SIRT3-transfected HCT116 cells. By disclosing\nthe first cell-active acetylating compounds capable of both global\nand target-selective post-translational acetylation of the catalytic\nlysine on kinases, our strategy could provide a useful chemical tool\nin kinase biology and drug discovery.